Document heading doi: 10.1016/S1995-7645(14)60232-7
G enetics study and transmission electron microscopy of pili in susceptible and resistant clinical isolates of M ycobacterium tuberculosis
Hossein Hosseini1, Abbas Ali Imani Fooladi1, Mohammad Arjomandzadegan2*, Navid Emami2, Hamid Bornasi2
1Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
2Tuberculosis and Pediatric Infectious Resaerch Center, Arak University of Medical Sciences, Arak, Iran
Asian Pacific Journal of Tropical Medicine
journal homepage:www.elsevier.com/locate/apjtm
*Corresponding author: Mohammad Arjomandzadegan, Tuberculosis and Pediatric Infectious Research Center and Department of Microbiology, Arak University of Medical Sciences, Arak, Iran.
E-mail: mmatinam81@yahoo.com; [email protected]
1. Introduction
Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis (M. tuberculosis) which is responsible for a huge number of deaths of humans throughout the history[1]. Despite the remarkable development and progress of technology, especially medical sciences and relative promotion of life styles, TB has still remained as a public health problem[2,3]. Within different parts of the world, the bacteria caused TB having a high distribution, and in a way that nine million human beings are infected with TB every year and relatively two million of them will die[4,5]. About one-third of the world population have been contaminated or at risk of TB[6]. However, more than 90%
of TB cases and deaths occur in developing countries.
Given the importance of this pathogen in the global health status, it is necessary to fight against and prevent it. Since prevention is always the most important step in fighting against a disease and lower costs will be imposed on individuals, exploring the ways that lead to preventive measures is of tremendous value. As a result, the emergence of drug-resistant strains and other items that might be caused by these pathogens will be prevented.
Pathogenic bacteria for mammalian and plant hosts have evolved virulence associated adherence appendages called pili that mediate directly or indirectly the interaction of bacteria with specific host target cells[7,8]. Pili are proteinaceous polymeric structures generally composed of a major repeating subunit or pilin and in some cases, a minor tip-associated adhesin subunit[9-11]. The hydrophobic nature of pili overcomes the repulsive forces between bacteria and eukaryotic cells. Due to their key roles in bacterial pathogenesis, pili are viewed as virulence factors and important targets for vaccine development[12].
ARTICLE INFO ABSTRACT
Keywords:
Mycobacterium tuberculosis Pili genes
Sequencing
Transmission electron microscopy
Objective:To study genetic bases and morphology of pili in Mycobacterium tuberculosis (M.
tuberculosis).
Methods: PCR and sequencing were used to investigate two related pili, Mtp and Flp genes in clinical isolates of M. tuberculosis. The primers were designed and PCR program were set for whole genes amplification. PCR products of the two genes from all isolates were sequenced by an applied biosystems apparatus and the results were analysed by online software. In the other hands, harvested cells from fresh cultures of isolates were undergoing specific sample preparation for sectional and negative staining for transmission electron microscopy.
Results: Electrophoresis revealed two specific bonds of 361 bp for Mtp and 150 bp for Flp genes and confirmed primer and PCR conditions designing. There were not any mutations in sequencing results of Mtp and Flp in comparison with reference sequence. Transmission electron microscopy examination revealed two distinct types of pili in the isolates as a bundle-forming pilus and rope-like pilus. From total investigated cells, 10% harbored pili in their structure.
Conclusions: Two genes of pili in all clinical isolates of M. tuberculosis were conserved and two morphological types of pili were detected. We proposed that by targeting pili proteins by a suitable inhibitor, it could affect the pathogenesis especially in resistant forms.
Article history:
Received 22May 2014
Received in revised form 6Jun 2014 Accepted 26Jun 2014
Available online 9Jul 2014
Contents lists available at ScienceDirect
Structurally, pili or fimbriae are straight or flexible filaments 5-7 nm wide and 2-3µm long. Many genes are required for pili biogenesis and encoded as the major pilin subunit, prepilin peptidase, chaperone, minor subunit, proteins involved in membrane translocation, nucleotide-binding proteins and channel-forming protein among others[7,8,13]. Pili have generally been associated with several biological activities such as agglutination of human and animal erythrocytes, bacterial adherence and colonization of mucosal surfaces. It is important to mention that there are many types of pili, and most microorganisms that produce them often have the capacity to produce more than one pili type. One pili type of M. tuberculosis, Mtp, with a molecular weight of 361 bp, is comprised of 4-kDa protein subunits encoded by the M. tuberculosis H37Rv predicted open reading frame, designated Rv3312A. A second pili morphotype that appeared as rope-like bundles was observed for M. tuberculosis which is known as Flp with a molecular weight of 150 bp. It is comprised of 6-kDa protein subunits encoded by the M. tuberculosis H37Rv predicted open reading frame, designated Rv3656c[14-
17]. Flp is the IV-type pili which is thin and flexible. It has a diameter of 6-7 nm and has a different pattern for monomers assemblage. This type of pili causes the bacteria to bind different surfaces. These pili are essential for the binding of bacteria to the epithelial cells. So, they play a key role in initiation and continuation of disease.
The type IV pilins have been further separated into two groups known as type IVA and IVB sub-families.
The subunits of IVA pili are found in various groups of human pathogens while the subunits of IVB pili are merely found in intestinal bacteria[8]. IVA (GC) is more resistant to proteolysis, heat, and destructive chemical materials, while IVB is more stable and its subunits are not assembled as strong as GC. Type IV is a prominent type of pili produced by many Gram-negative bacterial pathogens[8]. Type IV pili has been associated with virulence and other biological properties. These include twitching motility, bacterial aggregation, biofilm formation and development, colonization of host tissues and DNA transformation[8,18,19]. In the present research, after recognizing the genes producing pili within clinical strains of M. tuberculosis, their sequences were studied. Besides, the morphology of pili within the clinical strains observed by electron microscopy was focused.
2. Materials and methods
2.1. Sample collection and experimental method
The two basic genes producing pili were recognized as Flp and Mtp. The present research has focused on the presence of genes and their sequence within DNA in
clinical strains. For this purpose, 36DNA samples extracted from the sputum of TB patients were studied. The samples were composed of 18 samples of multidrug-resistant, 7 samples of extensively drug-resistant, and 11 sensitive samples. The Chelex method was applied to extract DNA
from bacteria samples. Briefly, in this method the positive sputum will be centrifuged with N-acetyl-L-cysteine treatment. The resulted specimen was transferred into a tube contains of 0.07 g chelex and 300µL of 1伊Tris-acetate-
EDTA buffer, then it was heated to a temperature of 95°C for 45 min. The tubes were centrifuged at 10000 r/min for 10 min. The supernatant contained of DNA was poured off and the specimen was centrifuged for two other times in order to have DNA without chelex, because chelex will cause disorder in PCR.
PCR was applied for approving the presence of the examined gene and sequencing method was used for determining the sequence of genes after extracting the
DNA. To this purpose, PCR was applied to the extracted
DNA of clinical strains of M. tuberculosis and the result of the PCR was prepared for sequencing. To this end, specific primers for amplification of the target genes were designed and used. At the first stage of the experiment, which is using PCR reaction, the test results must be investigated after the careful conduction of the reaction.
For this reason, the study was done by electrophoresis method using 1% agarose gel. Then, for determining the sequence of the nucleic acid within the mentioned genes, sequencing method was used. Within this method, the considered fragment consisted of the determined codons were amplified by PCR with specific primers. The results of the PCR were sequenced after purification.
2.2. Primer designing
According to the main objective of the research which is sequencing and analyzing pili genes, the primer of Mtp was designed by MEGA Software and ordered and the primer of Flp was ordered using the paper. To increase the accuracy of the operation of the designed primer, the following factors were considered and the best sequences were selected by decreasing and increasing the nucleoids:
length of the primer, percentage of the GC (between 45-
60), delta G prime, preventing the formation of hairpin structures, preventing sticky end, increasing the percent of C or A at the end of the three primers, increasing the AT percentage in 3’, preventing the formation of (3’) GATC…(5’) and also, (3’)GT…AC(5’) (Table 1), similarity in the percentage of GC within R and F, selecting a melting temperature between 65-70 °C and making a similar situation for both of the primers, and selecting annealing
temperature based on the melting temperature.
Table 1
The used primers within the experiment for carrying out PCR.
Primer name Primer (5'-3') Product size
(bp)
Flp-F (5’-ATGTTTCGTGTACTCGTGGCG-3’)
Flp-R (5’-CGCCGACACAATGGAATCCC-3’) 150
Mtp-F (5’-ACCGTCATCATATGTACCGGTTCG-3’)
Mtp-R (5’-CATCGGCTCGAGAGCGTAAATCTG-3’) 361
2.3. PCR
PCR reaction was carried out in the final volume of 25µL consisting of 2.5µL of purified DNA, 2.5µL of forward primer,
2.5 µL of revers primer, 12.5µL of master mix and finally,
5µL of water. The temperature plan was as follows: initial denaturation temperature of 94 °C for 5 min, denaturation temperature for each cycle is 94°C for 1 min, primer binding temperature is 59°C for 1 min, and elongation of primers for 45 seconds. Finally, PCR results were studied within 1% agarose gel.
2.4. Sequencing
The results of the PCR reaction were prepared for sequencing and transferred into the Source BioScience Company. The results were sent back after a few days.
2.5. Morphologic investigation of pili
The transmission of pili structure was observed using electron microscopy. To study the pili structure morphologically using transmission electron microscopy
(TEM), the specimens were cultured in Muler-Hinton broth as a first step. Then, the specimens were observed by electron microscopy and negative staining. For negative staining, approximately one drop of the specimens was placed onto a
300-mesh carbon formvar copper grid. Then, the parts of the needless specimen were wiped and negative staining was done by 1% uranyl acetate.
In order to do positive staining, the 2.5% glutaraldehyde solution within the phosphate buffer 0.1 mol/L was used as a primary fixation. Then, phosphate buffer 0.1 mol/L was used for washing and as a second fixation, ammonium tetroxide was used and then washing was repeated. Dehydration was done using 25%, 50%, 70%, 90%, and 100% ethanol concentrations for three times and each time for 10-15 min. Then the specimen was put in propylene oxide for two times (each time 30 min). Then it was placed in the resin and propylene oxide with the portions of 1:2, 1:1, and 2:1 for
2-3 h. Then it was put into pure resin and molded. Later, it was placed into an oven at 60°C for 2-3 d. Cutting was done using an ultra microtom machine Umo3 model of Richert Company, Austria. The slices were placed on a 300-mesh copper grid and investgated with TEM.
3. Results 3.1. PCR results
All of the 36 investigated strains selected for studying the two mentioned genes formed respective bond. It means that the 361 bp for Mtp and 150 bp for Flp are observed within agarose gel. Figure 1 shows the results of bond formation of Mtp for a number of specimens. Figure 2 shows the results of carrying out PCR for Flp for a number of specimens.
25e 90MDR 75MDR 571XDR 112MDR 42MDR124XDR30MDR 361 bp
Figure 1. PCR results of Mtp for a number of specimens.
28e 90MDR 75MDR 31e 25e 112MDR 1MDR 2XDR
150 bp
Figure 2. PCR results of Flp for a number of specimens.
3.2. Sequencing results
Sequencing results obtained from MEGA software indicated pili-related genes and confirmed PCR results. Figure 3 shows a part of Mtp sequence of specimens sent for sequencing.
As it is clear, this gene has the same sequence within all the specimens.
Figure 4 shows a part of Flp sequences. Within the first row, it is a part of the sequences of Flp and its following rows indicate sequencing results of a number of specimens.
Observing the true sequence of Flp confirms PCR results.
The results of electron microscopy are confirming and as it is shown in the pictures below, there observed both types of pili in M. tuberculosis. Figure 5 shows a bundle-forming pilus of Mtp while Figure 6 shows fiber type pili of Flp which is rope-like.
Figure 5. A bundle-forming pilus resulted from locus Rv3312A observable on the bacteria surface (40000伊 magnified).
Figure 6. A rope-like pilus resulted from locus Rv3656(40000伊 magnified).
4. Discussion
Tuberculosis is a chronic and infectious disease which is considered as one of the major cause of death in the world.
Tuberculosis is endemic in Iran and the world which is more prevalent within third-world countries[5,20]. The biggest reason for infection in such countries is malnutrition, however, genetic factors, age, and immunological conditions of the infected person should not be overlooked. As an example, it has been demonstrated that those animals which possess biocompatibility antigen human leukocyte antigen HLA-Bw15 may have a higher risk of disease[9,21]. Or people who are HIV-infected are at higher risk, because their immune systems are weaker[3]. Due to the increasing proliferation of drug-resistant TB and according to TB treatment method, providing some techniques for rapid detection of drug sensitivity or resistance of M. tuberculosis strains seems necessary[22]. Given the importance of this pathogen in the global health status, there is a clear necessity to fight against and prevent it. Since prevention is always the most important step in fighting against a disease and lower costs will be imposed on individuals, exploring the ways that lead to preventive measures is of tremendous value. As it was mentioned, due to their key roles in bacterial pathogenesis, pili are viewed as virulence factors and therefore as important targets for vaccine development.
Pili perform their roles by adhering to the lunge surfaces.
pili rv3312a
75MDR_mtpF
30MDR_mtpF 119MDR_mtpF 75MDR_mtpR 90MDR_mtpF 370XDR_mtpF 571XDR_mtpF 25e_mtpF
37e_mtpF 28e_mtpF 2XDR_mtpF 13N_mtpF
Figure 3. Sequencing results of the Mtp.
pili rv3656 75MDR_flpF
30MDR_flpF 119MDR_flpF 75MDR_flpR 90MDR_flpF 370XDR_flpF 571XDR_flpF 25e_flpF
37e_flpF 28e_flpF 2XDR_flpF 13N_flpF
Figure 4. Sequencing results of the Flp.
Therefore preventing reactions are of great importance. The results of the present research and other similar papers confirm the morphological presence of pili genes within the bacteria. So, further studies might be a step towards the creation of a vaccine against this pathogen. The present paper, genetically and morphologically, investigated the genes producing pili. Any disorder within the producing genes’ structures will cause inefficient units which lead to inefficiency in the pili operation. In 2007, for the first time, Alteri et al. reported that M. tuberculosis produced pili. Based on his findings using TEM and SEM electron microscopy, the presence of pili-like structures within the bacteria was confirmed and it was stated that this human pathogen was capable of producing two different types of pili according to the different conditions[23,24].
The results of the present research indicate that the genes are present within all the considered strains. These genes are away from any mutation and also, the findings of electron microscopy show morphologically the existence of pili on the surface of M. tuberculosis.
Conflict of interest statement
We declare that we have no conflict of interest.
References
[1] Lew JM, Kapopoulou A, Jones LM, Cole ST. TubercuList--10 years after. Tuberculosis (Edinb) 2011; 91: 1-7.
[2] Ojha AK, Hatfull GF. Biofilms of Mycobacterium tuberculosis:
new perspectives of an old pathogen. In: Cardona PJ, editor.
Understanding tuberculosis-deciphering the secret life of the bacilli. Reijek: Intech Open Access Publisher; 2012, p. 181-192. [3] Chen HY, Yu MC, Huang WL, Wu MH, Chang YL, Che CR, et
al. Molecular detection of rifabutin-susceptible Mycobacterium tuberculosis. J Clin Microbiol 2012; 50(6): 2085-2088.
[4] Jiao WW, Mokrousov I, Sun GZ, Li M, Liu JW, Narvskaya O, et al. Molecular characteristics of rifampin and isoniazid resistant Mycobacterium tuberculosis strains from Beijing, China. Chin Med J (Eng)2007; 120(9): 814-819.
[5] World Health Organization. Global tuberculosis report 2012. Geneva: World Health Organization; 2012. [Online] Available from: https://www.google.com/url?sa=t&rct=j&q=&esrc=s&so urce=web&cd=2&ved=0CCoQFjAB&url=http%3A%2F%2Fapps.
who.int%2Firis%2Fbitstream%2F10665%2F75938%2F1%2F9789241 564502_eng.pdf&ei=mLizU6PpL8yCqgbA8oLwCA&usg=AFQjC NF9pRMHb51lm4ZWJqh05L0HLxV3eg [Accessed on 6th March,
2013]
[6] Zignol M, Wright A, Jaramillo E, Nunn P, Raviglione MC. Patients with previously treated tuberculosis no longer
neglected. Clin Infect Dis 2007; 44: 61-64.
[7] Klemm P, Schembri MA. Bacterial adhesins: function and structure. Int J Med Microbiol 2000; 290: 27-35.
[8] Strom MS, Lory S. Structure-function and biogenesis of the type IV pili. Annu Rev Microbiol 1993; 47: 565-596.
[9] Islam MS, Richards JP, Ojha AK. Targeting drug tolerance in mycobacteria: a perspective from mycobacterial biofilms. Expert Rev Anti Infect Ther 2012; 10: 1055-1066.
[10] Velayati AA, Masjedi MR, Farnia P. Latent tuberculosis
(TB) bacilli: yes or no to preventive chemotherapy. Int J Mycobacteriol 2012; 1: 1-2.
[11] Zhavnerko G, Nikolaevich Poleschuyk N. Mycobacterium under AFM tip; advantages of polyelectrolyte modified substrate. Int J Mycobacteriol 2012; 1: 53-56.
[12] Finlay BB, Falkow S. Common themes in microbial pathogenicity revisited. Microbiol Mol Biol Rev 1997; 61: 136-169.
[13] Thanassi DG, Saulino ET, Hultgren SJ. The chaperone/usher pathway: a major terminal branch of the general secretory pathway. Curr Opin Microbiol 1998; 1: 223-231.
[14] Barksdale L, Kim KS. Mycobacterium. Bacteriol Rev 1977; 41:
217-372.
[15] Barrow WW, Ullom BP, Brennan PJ. Peptidoglycolipid nature of the superficial cell wall sheath of smooth-colony-forming mycobacteria. J Bacteriol 1980; 144: 814-822.
[16] Draper P, Rees RJ. Electron-transparent zone of mycobacteria may be a defence mechanism. Nature 1970; 228: 860-861. [17] Gordon J, White RG. Surface peptido-glycolipid filaments on
mycobacterium leprae. Clin Exp Immunol 1971; 9: 539-547. [18] Bieber D, Ramer SW, Wu CY, Murray WJ, Tobe T, Fernandez R,
et al. Type IV pili, transient bacterial aggregates, andvirulence of enteropathogenic Escherichia coli. Science 1998; 280: 2114-
2118.
[19] Skerker JM, Berg HC. Direct observation of extension and retraction of type IV pili. Proc Natl Acad Sci U S A 2001; 98:
6901-6904.
[20] Pang JM, Layre E, Sweet L, Sherrid A, Moody DB, Ojha A, et al. The polyketide Pks1 contributes to biofilm formation in Mycobacterium tuberculosis. J Bacteriol 2012; 194: 715-721. [21] Sasindran SJ, Torrelles JB. Mycobacterium tuberculosis infection
and inflammation: what is beneficial for the host and for the bacterium? Front Microbiol 2011; 2: 2.
[22] Velayati AA, Farnia P, Masjedi MR. Pili in totally drug resistant Mycobacterium tuberculosis (TDR-TB). Int J Mycobacteriol 2012; 1: 57-58.
[23] Alteri CJ, Xicohténcatl-Cortes J, Hess S, Caballero-Olín G, Girón JA, Friedman RL. Mycobacterium tuberculosis produces pili during human infection. Proc Natl Acad Sci U S A 2007; 104:
5145-5150.
[24] Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP4.0: discriminating signal peptides from transmembrane regions.
Nat Methods 2011; 8: 785-786.
